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Europaisches Patentamt
European Patent Office
Office eu rope en des brevets

0 610 937 A1

EUROPEAN PATENT APPLICATION

@ Date of publication of application:
17.08.94 Bulletin 94/33

@ Applicant: Packard Instrument Company, Inc.
2200 Warrenville Road
Downers Grove, IL 60515 (US)

@ Inventor: Schelrer, Winfrled
Wienergasse 46,
A- 2380 Perchtoldsdorf (AT)

© The present invention provides a method of increasing the duration of detectable photon emission of a
luciferase-luciferin reaction. The method provides a luciferase-luciferin reaction in which photon emission can be
detected for up to and including eight hours. A method of the present invention can also be used to detect the
presence of luciferase in biological samples. The present invention also provides a composition used in
detecting the presence of luciferase in biological samples.

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EP 0 610 937 A1

Field of the Invention

This invention relates generally to the stabilization of luciferase catalyzed luminescence. The invention
relates as well to compositions and methods for improving the lifetimes of the luminescence reaction. This
5 invention also includes assay kits for detection of an expressed luciferase gene used in reporter-gene
techniques.

Background of the Invention

io Luciferases are found in a wide variety of organisms including fireflies, photobacteria, jellyfish and many
others. Luciferases are enzymes which catalyze the production of light by oxidizing luciferin to oxyluciferin
in a process known generally as bioluminescence.

The production of photons by luciferase occurs through a two step reaction which consumes luciferin,
adenosine triphosphate (ATP) and O2. In the first step, luciferase catalyzes the formation of luciferyl

rs adenylate from luciferin and ATP. In this first step pyrophosphate is released and a Mg +2 cofactor or
another divalent cation is required for proper luciferase function. Upon formation, luciferyl adenylate remains
within the active site of luciferase. In the next step, luciferase oxidizes luciferyl adenylate to an electronically
excited oxyluciferin with the consumption of oxygen. Light production occurs when the electronically excited
oxyluciferin decays to the ground state oxyluciferin. The decay from the excited state to the ground state

20 occurs with the concomitant emission of a photon. The color of the light produced differs with the source of
the luciferase and appears to be determined by differences in the structure of the various luciferases.

Luciferases have recently become useful in reporter-gene technology. In this technique, a reporter
gene, such as a luciferase encoding polynucleotide is used as an indicator for the transcription and
translation of a gene in a cellular expression system. The reporter gene is operatively linked to a promoter

25 that is recognized by the cellular expression system. Other commonly used reporter genes include
galactosidase, and chloramphenicol acetyltransferase (CAT). In a typical reporter gene assay, a DNA vector
containing the reporter gene is transfected into a cell capable of expressing the reporter gene. After a
sufficient amount of time to allow for the expression of the reporter gene has passed, the cellular membrane
is disrupted to release the expressed gene product. The reagents necessary for the catalytic reaction of the

30 reporter gene are then added to the reaction solution and the enzymatic activity of the reporter gene is
determined. Alternatively, the cell can be disrupted in the presence of all reagents necessary for the
determination of the enzymatic activity of the reporter gene. If a ^-galactosidase encoding polynucleotide is
used as the reporter gene, the hydrolysis of a galactoside is determined. If a chloramphenicol acetyltrans-
ferase encoding polynucleotide is used as the reporter gene, the production of an acetylated chloram-

35 phenicol is determined. When luciferase is used as the reporter gene the photons produced from the
luciferase- luciferin reaction is measured.

A major problem in determining expression of a luciferase gene as a reporter gene is the short duration
of photon production- Typically, luciferase catalyzed photon production ceases within a few seconds. Means
for extending the period of photon production have been eagerly sought. Currently a commercially available

40 kit from the Promega Corporation (Madison, Wi) can extend the half-life of luciferase catalyzed photon
production to roughly five minutes. Nevertheless, for the measurement of large numbers of samples,
luciferase catalyzed photon production with a half-life of only five minutes is not a viable alternative. As
used herein, half-life is the time it takes for photon production to decrease by one half.

The present invention provides methods and compositions for increasing the duration of detectable

45 photon emission ofj luciferase-luciferin reaction.

Brief Summary of the Invention

In one aspect, the present invention provides a method for increasing the duration of detectable photon
50 emission of a luciferase-luciferin reaction. In one embodiment, a reaction mixture containing luciferase,
luciferin, ATP, and cofactors required for luciferase catalytic activity is mixed with a composition containing
adenosine monophosphate, a radical scavenger and a chelating agent to form an admixture. The photons
produced by the luciferase-luciferin reaction is then detected by measuring the luminescence of the
admixture. The luciferase catalyzed photon production can be detected for more than five minutes.
55 In a preferred embodiment, 100 ml of the admixture contains about 2.8 mg luciferin, about 110 mg
adenosine triphosphate (ATP) , cofactors necessary for luciferase catalytic activity, about 2.2 mg adenosine
monophosphate (AMP) , about 385 mg dithiothreitol (DTT) , and about 20 mg ethylenediaminetetraacetic and
(EDTA) .

EP 0 610 937 A1

In another embodiment, the amount of one of the components of the admixture can be varied while the
amounts of all other components remain unchanged. For example, in 100 ml of the admixture, the amount
of luciferin can be varied between about 0.2 to about 30 mg while maintaining the amount of ATP at about
110mg, AMP at about 2.2 mg, DTT at about 385 mg and EDTA at about 20 mg. Similarly, components can

5 be varied individually as follows: for 100 ml of the admixture, the amount of luciferin can be varied from
about 0.2 to about 30 mg; the amount of ATP can be varied from about 10 to about 300 mg; the amount of
AMP can be varied from about 0.2 to about 30 mg; the amount DTT can be varied from about 200 to about
2000 mg; and the amount EDTA can be varied from between about 10 to about 50 mg.

In another embodiment, the present invention contemplates a method for detecting the presence of

to luciferase in a biological sample. The biological sample suspected of containing luciferase is mixed with a
reaction mixture which contains luciferin, adenosine triphosphate, cof actors required for luciferase catalytic
activity, adenosine monophosphate, dithiothreitol, ethylenediaminetetraacetic acid, phenylacetic acid, oxalic
acid, and a detergent to form an admixture. The photons produced by the luciferase-luciferin reaction are
then detected by measuring the luminescence of the admixture.

75 In a preferred embodiment, the present invention contemplates an admixture for detecting the presence
of a luciferase in a biological sample. One hundred ml of the admixture contains about 2.8 mg luciferin,
about 110 mg adenosine triphosphate, about 2.2 mg adenosine monophosphate, about 385 mg dithioth-
reitol, about 20 mg ethylenediaminetetraacetic acid, about 4.5 mg phenylacetic acid, and about 0.85 mg
oxalic acid.

20 In another embodiment, the amount of one of the components of the admixture can be varied while the
amounts of all other components remain unchanged. For example, in 100 ml of the admixture, the amount
of luciferin can be varied between about 0.2 to about 30 mg while maintaining the amount of ATP at about
110mg, AMP at about 2.2 mg, DTT at about 385 mg, EDTA at about 20 mg, phenylacetic acid at about 4.5
mg, and oxalic acid at about 0.85 mg. Similarly, components can be varied individually as follows: for 100

25 ml of the admixture, the amount of luciferin can be varied from about 0.2 to about 30 mg; the amount of
ATP can be varied from about 10 to about 300 mg; the amount of AMP can be varied from about 0.2 to
about 30 mg; the amount DTT can be varied from about 200 to about 2000 mg; the amount of EDTA can be
varied from about 10 to about 50 mg; the amount of phenylacetic acid can be varied from about 1 to about
10 mg; and the amount of oxalic acid can be varied from about 0.2 to about 5 mg.

30 In another aspect, the present invention contemplates a composition used in detecting the presence of
luciferase in biological samples by detecting a emitted photon from a luciferase-luciferin reaction. One
hundred ml of this composition contains about 2.8 mg luciferin, about 110 mg adenosine triphosphate,
about 2.2 mg adenosine monophosphate, about 385 mg dithiothreitol, about 20 mg
ethylenediaminetetraacetic acid, about 4.5 mg phenylacetic acid, about 0.85 mg oxalic acid, and about 4 g

35 triton .

Brief Description of the Drawings

Figure 1 shows the effect of varying the AMP concentration on luciferase-luciferin light production.
40 Figure 2 shows the effect of varying DTT concentration on luciferase-luciferin light production.

Figure 3 shows the effect of varying both DTT and AMP concentration on luciferase-luciferin light
production.

Figure 4 shows the effect of various reagents to the luciferase-luciferin reaction.
Figure 5 shows the light production of a reporter-gene assay using a cloned luciferase.
45 Figure 6 shows that Compound A is either an unspecific inhibitor of the HIV-rev regulatory protein or is
toxic to the cells.

Figure 7 shows that Compound B is a specific inhibitor of the HIV-rev regulatory protein.
Detailed Description of the Invention

Luciferases catalyze the oxidation of luciferin with the concomitant emission of photons. The present
invention provides compositions and methods for increasing the duration of detectable photon emission of a
luciferase-luciferin reaction. The present invention provides a luciferase-luciferin reaction in which light
production can be detected for more than five minutes. In a preferred embodiment, the photon emission of
55 a luciferase-luciferin reaction can be detected for up to and including eight hours. In another embodiment,
detectable photon emission from a luciferase-luciferin reaction is linear for up to eight hours (see Figures 1 -
5).

EP 0 610 937 A1

In one embodiment, a reaction mixture containing luciferase, luciferin, ATP, and cofactors required for
luciferase catalytic activity is mixed with a composition containing adenosine monophosphate, a radical
scavenger and a chelating agent to form an admixture. The photons produced by the luciferase-luciferin
reaction are then detected by measuring the luminescence of the admixture. Luciferase catalyzed photon
5 emission as disclosed by the methods and compositions of the present invention can be detected for more
than five minutes. In a preferred embodiment, photon emission can be detected for more than thirty minutes
and for up to eight hours. In a preferred embodiment, photon emission decays linearly for up to eight hours
as shown in Figures 1-3.

In a preferred embodiment, the present invention provides a luciferase-luciferin reaction admixture. One
10 hundred ml of the admixture contains about 2.8 mg luciferin, about 110 mg adenosine triphosphate, about
2.2 mg adenosine monophosphate, about 385 mg dithiothreitol, and about 20 mg ethylenediaminetetraacetic
acid.

75 of luciferin can be varied between about 0.2 to about 30 mg while maintaining the amount of ATP at about
110mg, AMP at about 2.2 mg, DTT at about 385 mg and EDTA at about 20 mg. Similarly, components can
be varied individually as follows: for 100 ml of the admixture, the amount of luciferin can he varied from
about 0.2 to about 30 mg; the amount of ATP can be varied from about 10 to about 300 mg; the amount of
AMP can be varied from about 0.2 to about 30 mg; the amount DTT can be varied from about 200 to about

20 2000 mg; and the amount of EDTA can be varied from between about 10 to about 50 mg. As an example,
the present invention contemplates an admixture of the following composition: 100 ml of the admixture
contains about 2.8 mg luciferin, about 110 mg adenosine triphosphate, between about 0.2 to about 35 mg
adenosine monophosphate, about 385 mg dithiothreitol, and about 20 mg ethylenediaminetetraacetic acid.
Other admixtures in which only one component is varied in accordance with the limitations disclosed above

25 are contemplated.

In another aspect, the present invention contemplates a method for detecting the presence of luciferase
in a biological sample. The biological sample suspected of containing luciferase is mixed with a reaction
mixture which contains luciferin, adenosine triphosphate, cofactors required for luciferase catalytic activity,
adenosine monophosphate, dithiothreitol, ethylenediaminetetraacetic acid, phenylacetic acid, oxalic acid,

30 and a detergent to form an admixture. The photons produced by the luciferase-luciferin reaction is then
detected by measuring the luminescence of the admixture. A preferred biological sample is a cell that
produces luciferase. An exemplary detergent is triton N101. The present invention provides methods and
compositions in which the presence of luciferase in the biological sample can be detected for more than
five minutes by detection of the emitted photon. In a preferred embodiment, photon emission can be

35 detected for more than thirty minutes and for up to eight hours. In a preferred embodiment, photon
emission decays linearly for up to eight hours as shown in Figures 1-3.

In a preferred embodiment, the present invention contemplates an admixture for detecting the presence
of luciferase in a biological sample. One hundred ml of the admixture contains about 2.8 mg luciferin, about
110 mg adenosine triphosphate, about 2.2 mg adenosine monophosphate, about 385 mg dithiothreitol,

40 about 20 mg ethylenediaminetetraacetic acid, about 4.5 mg phenylacetic acid, about 0.85 mg oxalic acid,
and a detergent.

45 110mg, AMP at about 2.2 mg, DTT at about 385 mg, EDTA at about 20 mg, phenylacetic acid at about 4.5
mg, and oxalic acid at about 0.85 mg. Similarly, components can be varied individually as follows: for 100
ml of the admixture, the amount of luciferin can be varied from about 0.2 to about 30 mg; the amount of
ATP can be varied from about 10 to about 300 mg; the amount of AMP can be varied from about 0.2 to
about 30 mg; the amount DTT and be varied from about 200 to about 2000 mg; the amount of EDTA can

50 be varied from about 10 to about 50 mg; the amount of phenylacetic acid can be varied from about 1 to
about 10 mg; and the amount of oxalic acid can be varied from about 0.2 to about 5 mg.

In another aspect, the present invention contemplates a composition used in detecting the presence of
luciferase in biological samples by detecting emitted photons from a luciferase-luciferin reaction. One
hundred ml of this composition contains about 2.8 mg luciferin, about 110 mg adenosine triphosphate,

55 about 2.2 mg adenosine monophosphate, about 385 mg dithiothreitol, about 20 mg
ethylenediaminetetraacetic acid, about 4.5 mg phenylacetic acid, and about 0.85 mg oxalic acid.

In a more preferred embodiment, the present invention contemplates a reaction mixture for detecting
the presence of a luciferase in a biological sample, 100 ml of which contains about 2.8 mg luciferin, about

EP 0 610 937 A1

110 mg adenosine triphosphate, about 2.2 mg adenosine monophosphate, about 385 mg dithiothreitol,
about 20 mg ethylenediaminetetraacetic acid, about 4.5 mg phenylacetic acid, about 0.85 mg oxalic acid,
and a detergent. As an example, the present invention contemplates an admixture of the following
composition: 100 ml of the admixture contains about 2.8 mg luciferin, about 110 mg adenosine

5 triphosphate, about 2.2 mg adenosine monophosphate, between about 200 to 2000 mg dithiothreitol, about
20 mg ethylenediaminetetraacetic acid, about 4.5 mg phenylacetic acid, and about 0.85 mg oxalic acid.
Other admixtures in which only one component is varied as discussed above are contemplated.

As used herein, the term "luciferase" is an enzyme which catalyzes the oxidation of luciferin with the
concomitant emission of a photon. Luciferases can be isolated from biological specimens which produce

io luciferase or from a cell which has been transformed or transfected with a polynucleotide encoding for a
luciferase. It is within the skill of one of ordinary skill in the art to isolate a luciferase from a biological
specimen that produces luciferase. Similarly, means of transforming or transfecting a cell with a poly-
nucleotide that encodes for a luciferase are well known in the art.

One aspect of the invention provides a method for increasing the duration of detectable photon

;5 emission of a luciferase-luciferin reaction for more than thirty minutes by the addition of certain reagents. A
large number of substances which influence the luciferin-luciferase reaction including dithiothreitol, cytidine
nucleotides, AMP, pyrophosphate, coenzyme A, EDTA, protease inhibitors, and luciferase inhibitors includ-
ing luciferin anologs have been reported.

Decomposition and activation of luciferase decreases the lifetime of the luciferase-luciferin reaction by

20 inactivation of luciferase. In reporter gene techniques where cell lysates are used, the inactivation of
luciferase by endogenous proteases present in the cell lysate is a particular problem. The addition of
protease inhibitors such as phenylacetic acid (PAA), oxalic acid, monensine, acetyl phenylalanine, leupep-
tine, ammonium chloride, aprotinin and others prevent the degradation of luciferase by endogenous
proteases present in the biological sample. By slowing down or preventing the inactivation of luciferase,

25 luciferase catalyzed light production is lengthened. The above list is illustrative only and many other
protease inhibitors are well known in the art. The use of other protease inhibitors is contemplated by the
present invention.

The addition of luciferin analogs and other inhibitors of luciferase increase the lifetime of light
production by inhibiting luciferase. Care must be taken to ensure that the inhibitor binds to luciferase
30 reversibly. Analogs that bind irreversibly permanently inhibit luciferase. Exemplary reversible inhibitors of
luciferase include phenylbenzothiazol, 2-aminoethanol, benzothiazole, 2-hydroxyphenylbenzothiazole and
pyrophosphate.

Adenosine monophosphate (AMP) is a catalytic product of the luciferase-luciferin reaction. The addition
of AMP promotes the initial phase of the luciferase luciferin reaction. As shown in Figure 1. in the absence

35 of AMP, initial photon emission is low and maximum light production occurs nearly six hours after the
initiation of the luciferase-luciferin reaction. In contrast, when the concentration of AMP is between about 2.2
and about 35.2 mg/100 ml, maximum light production occurs immediately and decreases in a linear manner
for more than eight hours. Figure 1 shows the effect of varying the AMP concentration on luciferase-luciferin
light production. 100 ml of the admixture contained 110 mg adenosine triphosphate, 385 mg dithiothreitol,

40 2.8 mg luciferin, 20 mg ethylenediaminetetraacetic acid, 4 ml of a 10% triton N101 solution in H 2 0, 4.5 mg
phenylacetic acid, and 0.85 mg oxalic acid in 50 mM N-[2-hydroxyethyl] piperazine-N'-[2-ethanesulfonic
acid] (HEPES), pH, 7.8. The amount of AMP added to 100 ml of the assay solution was varied from about 0
to about 35.2 mg. With the addition of about 8.8 mg and about 35.2 mg of AMP, detectable photon
emission was linear between one and eight hours. The addition of 2.2 mg of AMP resulted in photon

45 emission which was linear for up to eight hours.

Thiol compounds such as dithiothreitol (DTT), dithioerythritol, glutathione and other well known reducing
agents are radical scavengers and increase the duration of detectable photon emission of a luciferase-
luciferin reaction. While the mechanism of thiol compound stabilization of the luciferase-luciferin reaction is
not well understood, these compounds probably function by limiting the availability of oxygen necessary in

50 the second step of the luciferase-luciferin reaction. At higher concentrations, DTT increases emission of
photons through an unknown mechanism. Figure 2 shows the effect of varying DTT concentration on
luciferase-luciferin light production, in Figure 2, 100 ml of the assay solution contained 2.2 mg adenosine
monophosphate, 110 mg adenosine triphosphate, 2.8 mg luciferin, 20 mg ethylenediaminetetraacetic acid, 4
ml of a 10% triton N101 solution in hfeO, 4.5 mg phenylacetic acid, and 0.85 mg oxalic acid in 50 mM N-[2-

55 hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid] (HEPES), pH 7.8. The amount of DTT in the admixture
varied from 193 to 770 mg. The addition of 385 and 770 mg of DTT resulted in detectable photon emission
which was linear for up than eight hours. When 193 mg of DTT is added, light production was reduced and
was linear between two and eight hours.

EP 0 610 937 A1

Figure 3 shows the effect of varying both DTT and AMP concentration on the duration of detectable
photon emission from a luciferase-luciferin reaction. 100 ml of the admixture contained 110 mg adenosine
triphosphate, 2.8 mg luciferin, 20 mg ethylenediaminetetraacetic acid, 4 ml of a 10% triton N101 solution in
H 2 0, 4.5 mg phenylacetic acid, and 0.85 mg oxalic acid, in 50 mM N-[2-hydroxyethyl] piperazine-N'-[2-
5 ethanesulfonic acid] (HEPES), pH, 7.8. The addition of 385 mg DTT and 2.2 mg AMP resulted in detectable
photon emission which was linear between 1 .75 and 8 hours. The addition of 770 mg DTT and 35 mg AMP
resulted in detectable photon emission which was linear for up to eight hours.

Chelating agents which bind metal ions can increase the duration of photon emission from a luciferase-
luciferin reaction by binding Mg +2 and other divalent cations including Ca+ 2 , Fe+ 2 , Mn+ 2 , Co 42 , and Zn+ 2 . In
w reporter gene techniques, the concentrations of divalent cations cannot be rigorously controlled because
whole cell lysates are used. Lysis of cells expressing a luciferase encoding gene release all of the
endogenous divalent cations including Mg +2 and Ca +2 into the luciferase-luciferin reaction admixture. The
addition of chelating agents effectively removes the released Mg 2+ and Ca +2 . Exemplary chelating agents
include ethylenediaminetetraacetic acid (EDTA), and ethylene glycol- bis (0-aminoethyl ether) N,N,N',N\-
;s tetracetic acid (EGTA). The use of other chelating agents are contemplated by the present invention.

The use of detergents to lyse cells suspected of expressing luciferase is well known in the art.
Exemplary anionic detergents include the series of triton detergents including triton N-101. The substitution
of other detergents, including both ionic and anionic detergents is within the skill of an ordinary artisan.

The use of reagents to maintain the pH of the luciferase-luciferin reaction solution is well known in the
20 art. An exemplary buffering reagent is N-[2-hydroxyethyl] pipera2ine-N*-[2-ethanesulfonic acid] (HEPES).
Many other buffering reagents are well known and are commercially available.

The present invention provides a combination of reagents which increase the duration of detectable
photon emission from a luciferase-luciferin reaction. Figure 4 shows the effect on the lifetime of the
luciferase-luciferin reaction in an admixture consisting of luciferase, luciferin, ATP, cof actors required for
25 luciferase catalytic activity and EDTA (20 mg/100 ml) in 50 mM HEPES and AMP, DTT or coenzyme A. In
all of the combinations shown in Figure 4, the detectable photon emission from a luciferase-luciferin
reaction lasts for at least for eight hours with the exception of a solution containing EDTA and 250 uM
coenzyme A in which photons can be detected for 7 hours.

The linearity in the decrease of photon emission during the life of a luciferase-luciferin reaction is an
30 important aspect of the present invention. Because photon emission is linear during the life of the
luciferase-luciferin reaction, initial luminescence can be calculated easily from the luminescence detected at
ay time during the life of the luciferase-luciferin reaction. From the calculated initial luminescence, the
concentration of the luciferase in the measured sample can be determined. For example, in the reporter
gene technique, samples suspected of expressing luciferase can be measured over a period of many hours
35 and the initial concentration of luciferase in the samples can be determined.

As discussed below in Examples 1 and 2 the present invention allows for the measurement of large
numbers of samples. Recently, instrumentation for measuring photon emission from 96 well microtiter
plates became available ( "TopCount Microplate Scintillation and Luminescence Counter" from the Packard
Instrument Company, Inc. of Downers Grove, Illinois). The light output from each 96 well plate takes roughly
40 ten minutes to measure. Thus with a linear emission of photons of eight hours, forty eight 96-well plates can
be easily measured. This results in the measurement of photon emission from 4608 individual samples.

Example 1 : Reporter-gene Assay Using a Luciferase cDNA

45 The reporter gene technique using a clone luciferase was used to demonstrate the increased duration
of the luciferase-luciferin reaction. The production of light from transiently transformed human T-cells
(Jurkat) was measured for eight hours. Jurkat cells in the logarithmic phase of growth were transformed by
the DEAE-dextran technique and incubated in a humidified incubator for 48 hours under assay conditions.
The plasmid vector contained a luciferase gene under the control of the EF promoter. After 48 hours, 100 ul

so of the cell suspension was mixed with 100 ul of PBS and 100 ul a 100 ml solution containing 2.2 mg
adenosine monophosphate, 110 mg adenosine triphosphate, 385 mg dithiothreitol, 2.8 mg luciferin, 20 mg
ethylenediaminetetraacetic acid, 4 ml of a 10% triton N101 solution in H 2 Q, 4.5 mg phenylacetic acid, 0.85
mg oxalic acid and 50 mM N-[2-hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid] (HEPES), pH 7.8. The
production of light was then measured for up to eight hours. The "high activity" curve (solid line) represents

55 light production from the transformation of Jurkat cells with 2 ug of the luciferase containing vector at a
concentration of 1 X 10 s cells/ml. The "low activity" curve represents light production from the same
experiment but with a cellular concentration of 50,000 cells/ml. The half life of the "high activity" sample
was found to be roughly 3j> hours and the half life of the "low activity" curve was found to be roughly 5

EP 0 610 937 A1

hours.

Example 2: High-capacity Luciferase Reporter Gene Assay for HIV-Rev-RRE-lnteraction

5 A transient transfection assay using the DEAE-dextran technique for T-cell lines was established to
screen for inhibitors of the HIV-rev regulatory protein (RRE) . Two expression vectors, one for the HIV-1
gene (pEF-cRev) and the other for a luciferase reporter gene (pCMV-Luc/RRE) are cotransfected in the
Jurkat cell line (lymphoblastoid ceils). RRE is expressed together with Luciferase if a certain level of
functional rev protein has been accumulated within a cell. The assay mimics the natural action of rev. Rev

jo transports unspliced or singly spliced RRE-containing mRNA out of the nucleus to the translation machinery
in the cytoplasm. If a certain threshold level of rev protein accumulates in the nucleus, rev binds to RRE
containing mRNA, multimerizes and mediates transport of this mRNA into a cellular pathway bypassing the
cellular splicing machinery. This is accomplished by binding of the Rev/RRE complex to a cellular factor,
translation initiation factor elF-5a, of the preribosomes formed within the nucleoli. Inhibitors of any of these

75 steps in the mode of action of rev is detectable in this reporter gene assay.

The assay conditions have been optimized to obtain a stimulation of luciferase expression by rev to
approx. 20-100 fold over background within 20 hours of transfection. The basal luciferase expression as
determined in control transfections with an identical expression vector carrying the rev gene in antisense
orientation to the promoter (pEF-AScRev) is usually below 5% of the rev-induced expression. The toxicity of

20 candidate compounds is determined by a parallel transfection assay. In this assay, a rev-independent
luciferase construct (pCMV-LUC) is cotransfected with the rev-expression vector )pEF-cRev) to keep
conditions similar to the rev-assay. This toxicity assay also identifies compounds which are inhibitors of the
luciferase enzyme or to transcription factors binding to the CMV-promoter. Therefore, the toxicity assay is
also capable of acting as a specificity control.

25 The assay has been adapted to microplate technology and can be automated. While detectors to
measure multiple samples have been known for some time, modern single photon measurement techniques
with an extraordinarily high sensitivity for detection of bioluminescence signals from microplates came to
the market recently. The stabilization of the bioluminescence signal from a half-life of a few minutes to more
than 4 hours allows for the automation of the assay by using the newly available microplate single photon

30 counters. A cDNA encoding for a luciferase is therefore an ideal reporter gene. The use of a luciferase is
applicable to many other mechanism-based cellular test systems for pharmaceutical, toxicological and other
screening systems.

Jurkat T-cell suspension cultures, stock 1722 are split daily with a dilution of at least 1:2. Final cell
density should not exceed 6x1 0 5 cells/ml to keep the cells in the logarithmic growth phase. Cells can be
35 seeded at a density 0.5x1 0 5 /ml or lower for maintaining stock cultures. Culturing in roller bottles (approx. 10
rpm) is the preferred culture method.

The following buffers and reagents are used:

- Dulbecco's PBS (phosphate buffered saline)

- DEAE-Dextran (MW ca. 500.000), PHARMACIA # 17-0350-00 solved at 10 mg/ml in PBS and filtered
40 through 0.2 urn Nalgene filters.

- Assay medium: RPMI 1640 without phenol red supplemented with:

+ 10% FCS inactivated
+ 1% v/vHEPES buffer (1 M)
+ 1% Penicillin/Streptomycin, GIBCO 043-05070
45 + 0.1% Gentamicin, GIBCO 043-05750

+ 1% Chloroquine (10mM = 5.52 mg/ml in PBS), Sigma #C6628

- Wash medium: RPMI 1640 without phenol red supplemented with:

+ 1% v/v HEPES buffer (1 M)
+ 1% Penicillin/Streptomycin, GIBCO 043-05070
so + 0.1% Gentamicin, GIBCO 043-05750

- DNA preparations, purified by two cycles of CsCI gradient centrifugation and stored frozen in aliquots
at -20" C (dissolved at 1 mg/ml in H 2 0)

pCMV-Luc/RRE DNA, # 18xx
pEF-cREV DNA, # 21 xx
55 pEF-AScREV DNA, # 22xx

pCMV-Luc DNA, # 3xx

- luciferase-substrate solution:

2.2 mg AMP (Sigma A1877)

EP 0 610 937 A1

110 mg ATP (Sigma A5394)
385 mg Dithiothreitol

280 ul Luciferin (10 mg/ml in H 2 0), Sigma L5256 dissolved in 100 ml buffer consisting of:
5 ml HEPES(1M)
5 20 mg EDTA (Titriplex III)

4 ml TRITON N101 (10% in H 2 0)

4.5 mg Phenylacetic acid. Sigma No. P4514 0.85 mg Oxalic acid, Sigma No. 07626
mg adjusted to 7.8 (2N NaOH, ca 1.1 ml)

- Microplates:

io Packard CulturPlates 6005180 with adhesive seals

- Cellfilter Falcon #2340
The assay protocol is as follows:

Preparation of Dilutions:

Pure substances CHC (500uM in DMSO saturated H 2 Q)) 1:50 final dilution:

- Medium = RPMI 1640 plus additives (see above)

- Predilution: 8 ul sample plus 192 ul assay medium (Microplate)

20 - Add 50 ul predilution each in parallel positions to 3 white Packard CulturPlates (B1 through H12)

- Blank: 50 ul medium (A1 through A12)

- Plates are kept in a C02-cabinet until addition of coils

Broth of Actinomycetes at 1:200, Fungi at 1:100 and Bacteria at 1:100 final dilutions :

- Predilution: 8 ul sample plus 392 ul assay medium = 1:50 (Titertek racks), for bacteria and fungi
8 ul sample plus 792 ul assay medium = 1:100 (Titertek racks), for actinomycetes

- Add 150 ul of predilution each in 3 parallels to white Packard CulturPlates (B1 to H12)

- Blank: 50 ul assay medium (A1 to A12)

30 - Plates are kept in a C0 2 -cabinet until addition of cells

Designation of control wells in the primary screen:

A1, A2

plate control

medium only, no cells

A3 to A10

'high' control

pCMV-Luc/RRE + pEF-cREV

A11, A12

plate control

medium only, no cells

Secondary screening (validation) of the positive/toxic hits (1:50 to 1:6400 final dilution for substances):

- Predilution: 20 ul sample plus 480 ul assay medium (Titertek racks)

- Add 100 ul predilution in duplicates to the wells of rows A (3 to 12) to two Packard CulturPlates in
45 parallel

- Add 50 ul of assay medium to all the remaining wells

- Dilute columns 3 to 1 2 by serially transferring 50 ul and discarding the rest after row H.
Preparation of Cells and Transfection Procedure:

- Cells as described above, are centrifuged (200 g, 10 min) and washed twice with prewarmed wash
medium.

- Cells are suspended in prewarmed PBS and counted in a hemacytometer; the cell density is adjusted
to 4x10 6 cells/ml.

55 - DNA-solutions are prepared in prewarmed PBS (2.5 ml/plate, 25 ul/well to be tested):

- Add 25 ul DEAE/Dextran pr ml PBS.

- Add DNA:

- For REV/RRE : 0.60 Ul/ml pCMV-Luc/RRE # 18xx and 1 ul/ml pEF-cREV # 21xx.

EP 0 610 937 A1

- For negative controls : 0.60 ul/ml pCMV-Luc/RRE # 18xx and 1 ul/ml AScREV # 22xx.

- For toxicity/unspecific inhibition : 0.60 ul/ml pCMV-Luc # 3xx and 1 ul/ml pEF-cREV # 21 xx.

- For toxicity/handling control : 0.60 ul/ml pCMV-Luc # 3xx and 1 ul/ml pEF-AScREV # 22xx.

- Add an equal volume of cell suspension (4x1 0 6 cells/ml) to each of the DNA-solutions.

5 - The DNA/cell suspension is incubated for 10 min. at 37 \ shaken gently by hand and incubated for
another 10 min.

- An equal volume of prewarmed assay medium is added, cells are shaken gently by hand and
incubated for another 10 min.

- The pelleted cells are resuspended in prewarmed RPMI 1640 plus additives (assay medium) and
70 adjusted to a final concentration of 1x10 s cells/ml.

- Cells are sucked once through a syringe and filtered through a cell filter immediately before being
dispensed to the plates.

- 50 ul of cell suspension is added to each of the respective wells:
Secondary Jurkat-Rev Assay :

75 - 'High' control and substance dilutions: pCMV-luc/RRE + pEF-cRev in wells A1-H1

- Blanks: 50 ul medium in wells A2-D2

- 'Low* control: pCMNMuc/RRE + pEF-AScRev in wells E2-H2
Secondary Jurkat-Toxicity Assay :

- 'High' control and substance dilutions: pCMV-luc + pEF-cRev in wells A1-H1
20 - Blanks: 50 ul medium in wells A2-D2

- 'Handling' control: pCMV-luc + pEF-AScRev in wells E2-H2

- Plates are shaken using a microplate-shaker all stored in the incubator at 37* C in a 5% CO2
atmosphere for 16 - 24 hours.

25 Measurement of Luciferase Activity

- After incubation, 100 ul of luciferase substrate solution, to be prepared freshly every day, is added to
each well.

- Plates are shaken using a microplate shaker for a few seconds and sealed with an adhesive seal (do
30 NOT use heat-seals).

- Plates are counted in a Packard TopCount Microplate Scintillation and Luminescence Counter" or an
equivalent instrument after a count delay of 2 min. for 0.15 min. per well. The temperature of the
counting chamber as well as the plate stack should not exceed 22 • C, otherwise the half life of the
light emission can drop below 4 hours.

35 - The REV-Luc/RRE ('high'-control) - and the Luc (toxicity-control) - background corrected valued
should be at last 2000 cps, the As REV-Luc/RRE ("low "-control) should not exceed 100 cps.

Results

40 Figures 6 and 7 show the results of a large scale compound screening test for compounds which inhibit
HIV-rev regulatory protein (RRE). Jurkat cells are co-transfected with two plasmids, pEF-cRev and either
pCMV-Luc/RRE or pCMV-Luc. The closed square represents cells which have been transfected with pEF-
cREV and pCMV-Luc/RRE. The open square represents cells which have been transfected with pEF-cREV
and pCMV-Luc. If a candidate compound inhibits RRE, cells transfected with pEF-cREV ad pCMV-Luc/RRE

45 should show a decrease in light production from the luciferase-luciferin reaction compared to cells
transfected with pEF-cREV and pCMV-Luc. If a candidate compound does not inhibit RRE, then light
production from the luciferase-luciferin reaction should remain unchanged whether cells are transfected with
pCMV-Luc/RRE or pCMV-Luc.

Figure 6 shows the effect of Compound A on RRE. The inhibition curves obtained from cells co-

50 transfected with pEF-cRev and either pCMV-Luc/RRE (closed square) or with pCMV-Luc (open square) is
not dramatically different. Compound 6 therefore does not appear to be an inhibitor of RRE. Alternatively,
the toxicity of Compound A may prematurly kill the transfected cells before meaningful data can be
obtained.

Figure 7 shows the effect of Compound B on RRE. The inhibition curves obtained from cells co-
55 transfected with pEF-cRev and either pCMV-Luc/RRE (closed square) or with pCMV-Luc (open square) is
dramatically different. There is a significant difference in inhibition of RRE between cells transfected with
pCMV-Luc/RRE and cells transfected with pCMV-Luc. In cells transfected with pCMV-Luc/RRE, about 0.1
uM Compound B results in 50% inhibition. In cells transfected with pCMV-Luc, Compound B must be

EP 0 610 937 A1

present in concentrations greater than 10 uM to obtain 50% inhibition (data not shown).
Claims

5 1. A method for increasing the duration of detectable photon emission of a luciferase-luciferin reaction
mixture containing luciferase, luciferin adenosine triphosphate, and cofactors necessary for luciferase
catalytic activity, the method comprising mixing the reaction mixture with a composition comprising
adenosine monophosphate, a free radical scavenger and a chelating agent to form a admixture.

io 2. The method of Claim 1 , wherein the free radical scavenger is dithiothreitol.

3. The method of Claim 1 , wherein the chelating agent is ethylenediaminetetraacetic acid.

4. The method of Claim 1, wherein 100 ml of the admixture contains about 2.8 mg luciferin, about 110 mg
75 adenosine triphosphate, about 2.2 mg adenosine monophosphate, about 385 mg dithiothreitol, and

about 20 mg ethylenediaminetetraacetic acid.

5. The method of Claim 1, wherein 100 ml of the admixture contains between about 0.2 to about 30 mg
luciferin, about 110 mg adenosine triphosphate, about 2.2 mg adenosine monophosphate, about 385

20 mg dithiothreitol, and about 20 mg ethylenediaminetetraacetic acid.

6. The method of Claim 1, wherein 100 ml of the admixture contains about 2.8 mg luciferin, between 10 to
about 300 mg adenosine triphosphate, about 2.2 mg adenosine monophosphate, about 385 mg
dithiothreitol, and about 20 mg ethylenediaminetetraacetic acid.

7. The method of Claim 1, wherein 100 ml of the admixture contains about 2.8 mg luciferin, about 110 mg
adenosine triphosphate, between about 0.2 to about 35 mg adenosine monophosphate, about 385 mg
dithiothreitol, and about 20 mg ethylenendiaminetetraacetic acid.

30 a The method of Claim 1, wherein 100 ml of the admixture contains about 2.8 mg luciferin, about 110 mg
adenosine triphosphate, about 2.2. mg adenosine monophosphate, between about 200 to 2000 mg
dithiothreitol, and about 20 mg ethylenediaminetetraacetic acid.

9. The method of Claim 1, wherein 100 ml of the admixture contains about 2.8 mg luciferin, about 110 mg
35 adenosine triphosphate, about 2.2 mg adenosine monophosphate, about 385 mg dithiothreitol, and

between about 10 to 50 mg ethylenediaminetetraacetic acid.

10. A method for detecting the presence of luciferase in a biological sample comprising:

(a) mixing a sample suspected of containing luciferase with a reaction mixture containing luciferin,
40 adenosine triphosphate, cofactors necessary for luciferase catalytic activity, adenosine mon-
ophosphate, a protease inhibitor, a free radio scavenger, a chelating agent, and a detergent to form
an admixture and

(b) measuring the luminescence.

45 11. The method of Claim 10, wherein the free radical scavenger is dithiothreitol.

12. The method of Claim 10, wherein the chelating agent is ethylenediaminetetraacetic acid.

13. The method of Claim 10, wherein the protease inhibitor is phenylacetic acid or oxalic acid.

14. The method of claim H), wherein the detergent is triton N-101.

15. The method of Claim 10, wherein 100 ml of the admixture contains about 2.8 mg luciferin, about 110
mg adenosine triphosphate, about 2.2 mg adenosine monophosphate, about 385 mg dithiothreitol,

55 about 20 mg ethylenediaminetetraacetic acid, about 4.5 mg phenylacetic acid, and about 0.85 mg
oxalic acid.

EP 0 610 937 A1

16. The method of Claim 10, wherein 100 ml of the admixture contains about 2.8 mg luciferin, about 110
mg adenosine triphosphate, about 2.2 mg adenosine monophosphate, about 385 mg dithiothreitol,
about 20 mg ethylenediaminetetraacetic acid, between about 1 to about 10 mg phenylacetic acid, and
about 0.85 mg oxalic acid.

17. The method of Claim 10, wherein 100 ml of the admixture contains about 2.8 mg luciferin, about 110
mg adenosine triphsophate, about 2.2 mg adenosine monophosphate, about 385 mg dithiothreitol,
about 20 mg ethylenediaminetetraacetic acid, about 4.5 phenylacetic acid, and between about 0.2 to
about 5 mg oxalic acid.

18. The method of claim 10, wherein the biological sample comprises a cell that produces luciferase.

19. A composition used in detecting the presence of luciferase in biological samples wherein 100 ml of the
composition contains about 2.8 mg luciferin, about 110 mg adenosine triphosphate, about 2.2 mg

75 adenosine monophosphate, about 385 mg dithiothreitol, about 20 mg ethylenediaminetetraacetic acid,
about 4.5 mg phenylacetic acid, about 0.85 mg oxalic acid, and about 4 g triton .

EP 0 610 937 A1

i ii lf li

EP 0 610 937 A1

European Patent
Office

EUROPEAN SEARCH REPORT

Application Number

EP 94 10 2080

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